Gene regulation in prokaryotes Flashcards
Does the larger the organism mean the larger the genome size?
yes
generally what has a larger genome, eukaryotes or prokaryotes?
eukaryotes
Is prokaryotic DNA exposed?
yes
Tell me about E. Coli’s genome, number of nucleotide pairs and how many proteins is approx. codes for?
- consists of a singular circular DNA molecule
- 4.6x106 nucleotide pairs
- encodes for approx. 43,000 proteins
generally, how many proteins do prokaryotic genomes encode?
1000-6000 proteins
Are prokaryotic genomes, diploid or haploid?
They only contain one copy of each gene
they are haploid
How is DNA stored in prokaryotes?
Whats an exception of this?
The DNA is compressed and packaged into a small space via supercoiling
The exception of a species within archaea which doesnt do this
Tell me about transcription and translation in prokaryotes?
The chromosomes are localised in the cytoplasm and transcription and translation takes place simultaneously
Whats a Nucleoside?
A nitrogenous base attached to a 5-carbon sugar
Whats a nucleotide?
A nucleoside attached to a phosphate group
How many H bonds between AT and CG?
CG has 3 H bonds
AT has 2 H bonds
What is a purine made from?
Purine = pyrimidine + imidazole
Whats an imidazole ?
A histamine side chain
Complete the label in the following structure
What are the purines and pyrimidines ?
Whats the structure of the purines and pyrimidines?
What are nucleoside analogues used for?
How are these components activated?
Why are they used?
nucleoside analogues are used as antiviral and anticancer agents
These components are activated in the cell by being converted to nucleotides
They are administered as nucleosides since charged nucleotides cannot easily cross cell membranes
Tell me the size of the following in DNA (in nm)…
- Diameter
- Major groove
- Minor groove
- Length of one turn
- DNA diameter= 2 nm
- Major groove in double strand is 2.2 nm = 22 Å
- Minor groove in double strand is 1.2 nm = 12 Å
- Length of one turn= 3.4 nm
What does the centra dogma of molecular biology deal with?
What does it state?
‘The central dogma of molecular biology deals with the detailed residue-by-residue transfer of sequential information’
It states that such information cannot be transferred back from protein to either protein or nucleic acid.’- Francis crick
Or more simply- DNA makes RNA and RNA makes protein
If you have more proteins at a higher level, do they make more or less mRNA?
They make more mRNA
What are the three common types of RNA and what is their role?
There are three common types of RNA:
1. mRNA or messenger RNA: This is the one that transcribes the genetic code from DNA into a form that is used to make protein. mRNA carries the message from the nucleus to the cytoplasm of the cell.
2. rRNA or ribosomal RNA: Forms structural elements of the ribosome that direct the translation of mRNA into protein.
3. tRNA or transfer RNA: tRNA transfers amino acids to the ribosome
is there a base 0 in DNA/RNA?
Which direction is upstream/ downstream?
no
if Uracil was present in DNA why would this cause issues?
If uracil was present in DNA then it would generate more errors due to the ability to bind with more
Which strand in DNA is copied?
What is T replaced with?
Why can T only pair with A?
What can U pair with why does this lead to less mutations?
It is the non-coding or template strand that is copied and thymine (T) is replaced with uracil (U)
Due to the methyl group, T can only base pair with A
while U can base pair with others – better DNA fidelity therefore less mutation
What direction does the DNA coding sequence always run in?
5’ to 3’
However, the same DNA section can contain additional coding regions on the reverse strand again running in the 5’-to-3’ direction
How are the coding regions of DNA defined?
Define where coding starts as could start from any point which would lead to production of lots of different proteins
A reading frame define the coding regions
Whats a reading frame?
A reading frame is a set of consecutive, non-overlapping triplet nucleotides that equates to amino acids or stop signals during translation
They are called codons.
A gene is a fuctional unit which has…
- *1. Promotor
2. Operator**
3. Coding sequence
4. Terminator
What occurs at each of these?
- Promotor: Where the RNA polymerase binds
- Operator: site next to promotor where the regulatory protein binds
- coding sequence: for protein or RNA flanked by a start and stop codon
- terminator: where RNA polymerase ends transcription
What two features are present in the prokaryotic sequence?
Whats the base number of each?
What variation could occur?
-35 region and Pribnow box (-10)
Distance between the two stays the same but the number of bases can vary
What are the three phases of transcription and what happens in each?
1. Initiation: binding of RNAP holoenzyme (core polymerase + sigma () factor
2. Elongation: open complex formation (transcription bubble), DNA unwinding forming single strands within the active site
3. Termination: a termination signal in the DNA forms an RNA hairpin in the emerging transcript that causes the polymerase to be released
Tell me about the structure of RNA polymerase ?
What is each subunit used for?
- The bacterial core RNA polymerase consists of four proteins (alpha ßß’w) that forms a complex of five subunits (alpha2 ßß’w)
- The alpha-dimer plays a regulatory role during initiation
- w subunit is required for assembly but not required after that
- The ß and ß’ subunits form the active site and make all of the contacts to the nucleic acids
- The RNA polymerase holoenzyme contains an additional σ factor that is involved in initiation
- Omega is required for assembly, isn’t in name but it is present. Binds to the promotor regions on the DNA
What are E.coli’s sigma factors?
The gene of each?
The use of each?
Whats the HTH and what does it bind to?
HTH – Helix-Turn-Helix structural motif that binds the major groove of DNA
Tell me about the σ70 sigma factor of E.coli?
σ70 the “housekeeping” sigma factor
transcribes most genes in growing cells. Every cell has a “housekeeping” sigma factor that keeps essential genes and pathways operating
Label this RNA polymerase
Label this prokaryotic transcription cycle
Tell me the stages to the initiation of prokaryotic transcription…
What does this stage ensure?
- The sigma factor associates with the core RNA polymerase
- The holoenzyme searches for a promoter site
- The sigma factor recognises the appropriate consensus sequences
- It promotes the local unwinding of the DNA (~14 bps) to expose the template strand for copying. This starts at the Pribnow box (TATAAT) with the relatively low number of H-bonds. So easy to pull apart
- The RNA Polymerase cannot start forming full length transcript until it releases the strong binding to the promoter.
- Repetitive synthesis and release of short nascent RNAs by RNAP (abortive initiation)
- This always happens before steady-state transcription
- ~95 % of the total RNA contains RNA products ranging between 2-16 nts in length
- Dissociation of sigma factors
- Promoter clearance
- Ensures proper orientation of DNA and RNA polymerase complex for entering into the elongation stage
Tell me the features of DNA dependent RNA polymerase catalysis reaction
(Elongation)
- Direction of synthesis is 5’ to 3’
- No primer required
- Template required
What does RNA polymerase catalyse the formation of and to form what?
(elongation)
RNA polymerases catalyse the formation of the phosphodiester bonds that link the nucleotides together to form a linear chain of RNA in the 5’ to 3’ direction
What does one round of polymerisation yield?
(elongation)
A pyrophosphate
What are the substrates of an RNA polymerase enzymatic reaction?
(elongation)
The substrates are ribonucleotides triphosphates (ATP, CTP, UTP and GTP)
What are the two termination mechanisms in prokaryotic transcription?
- Rho dependent
- Rho independent
Tell me about the Rho independent mechanism
- Occurs when the termination signal is in the sequence of the RNA transcript itself.
- Hairpin forms followed by a stretch of uracil nucleotides
- Stem loop structure causes the RNAP to pause
- dA:U base pairs more unstable (fewer H-bonds) and complex dissociates
Whats a Rho factor?
Where does it attach and when?
A ring like structure which binds around to mRNA as it is being made
It attaches to the utilisation (rut) site on the mRNA
How does Rho factor work?
It moves along the mRNA in the same direction of the polymerase
As hairpin forms, RNAP pauses allowing Rho to catch up
Once in contact with RNAP, Rho pulls the mRNA out of the polymerase
In elongation, what is removed to form a new phosphate bond ?
PPi removed
Is it DNA or proteins that define us?
- A living organism is not its DNA
Human and chimps 99 % identical. Unrelated humans 99.9 %
- Yes, DNA is the blueprint, but it is the proteins and what they do that defines what and organism becomes and how it behaves
- So, what proteins are expressed and when, defines an organism
- We know that generally, the greater the level of mRNA transcript the more that that target protein is expressed
Can the expression of numerous genes be controlled simultaneously?
yes
Whats an operon?
Operon: a set of genes that are transcribed from the same promotor and controlled by the same operator site and regulatory proteins
Tell me about regulation of genes
Regulation: a set of genes (and/or operons) expressed from separate promoter sites but controlled by the same regulatory molecule. Global regulons may coordinate expression of many genes and operons, and may induce some, but repress others.
What the presence of an operon allow?
The coordinated regulation and gene expression of multiple genes at the same time
The RNA transcript made from an operon codes for what?
Codes for the multiple proteins (polycistronic RNA)
What can operators be in terms of direction?
Upstream, downstream or overlapping with the promotor
Regulatory proteins that bind with the operator can influence what?
The access of RNA polymerase to the promotor, thereby, affecting the rate of transcription initiation
Tell me about the control at the level of mRNA transcript?
- Attenuation of transcriptional elongation
- Codon usage
- Controlling access of RNAP
Repressors
Activators
- Riboswitch
- Packaging of the DNA as supercoils to prevent access
The rate at which ribosomes makes proteins in elongations depends on what?
The availability of the charged tRNA with the appropriate amino acid i.e. tRNATrp
What does the trp operon code for?
Genes involved in the synthesis of Trp
What happens when the avaliability of Trp is low?
The prokaryote will want the operon active, producing the enzymes that make more Trp and vice versa
Tell me about the Trp operon in the presence of high and low [Trp]
Whats the preferred codon to produce ?
The one with the highest ratio
e.g. Leucine CUG
How many possibilities are there with DNA and codons?
There are 4 nucleotides used in DNA. A triplicate of bases, the codon, codes for a single amino acid, therefore, there are 43 = 64 possibilities.
The diagram shows the relative frequency of codons within two genes in the same organism
- This is used in laboratories to increase protein expression levels
- It can have major effects in non-coding regions altering the RNA secondary structure
- Effect on the speed of the ribosome with faster rates along transcripts with higher codon adaption to tRNA pools
- Is also used as a means of controlling protein folding as mRNA translation is controlled both spatially and in time.
What are transcription factors?
Whats their role?
(sequence-specific DNA-binding factor) are proteins that control the rate of transcription
These proteins bind to the operator sites and either enhance or inhibit the binding of RNAP at the promoter site
What are the two parts of transcription factors?
(1) That binds to DNA
(2) That binds to the signal (in many cases this is a small molecule)
Whats the Trp transcription factor?
trpR= Trp repressor and it is the transcription factor that binds to Trp amino acids
Is the unbound trpR a suitable conformaton to bind to DNA?
no
what happens with trpR when [Trp] is high?
trpR binds to Trp and undergoes a conformational change that allows it to bind to the operator site
What does it mean when trpR is bound to the operator site?
It prevents RNA polymerase from starting transcription by physically blocking it
trpR and Trp operon
Although glucose is the preferred carbon source, prokaryotes like E.coli can metabolise other sources of energy such as lactose. How is this done?
by activating the lac operon if glucose is not available
What are the 3 structural genes that the lac operon consists of and what is the role of each?
1. lacZ - the enzyme beta-galactosidase which cleaves the disaccharide lactose into glucose and galactose
2. lacY – the membrane protein transporter that allows more lactose to enter the cell
3. lacA - beta-galactosidase transacetylase whose role is unknown
The lac operons role
What is the lac operon regulated by?
By a transcription factor called catabolite activator protein (CAP)
The lac operon systems role is to sense the overall energy needs of the organism and respond appropriately.
What does E. coli use to do this?
The internal metabolite that E. coli uses 3’,5’-cyclic adenosine monophosphate (cyclic AMP or cAMP)
What is cAMP derived from?
its derived intracelluarly from ATP
Tell me how each of the following effect transcription and cAMP concentration levels?
- [Glucose] is high
- [Glucose] is low
- When [glucose]i is high, [cAMP]i is low leads to low transcription
- When [glucose]i is low, [cAMP]i is high leads to high transcription
How many binding sites does the lac repressor protein have?
4
Tell me what happens in the following situations?
a) - lactose, +glucose, low cAMP
b) + lactose, + glucose, low cAMP
c) + lactose, - glucose, high cAMP
What activates the lac operon by CAP ?
How?
CTD
- The CTD interacts to the CAP when attached to the C terminal domain
- The alpha-subunits of the RNAP are composed of two domains the N-terminal and C-terminal domains (NTD and CTD, respectively).
- CAP is recognised by the CTDs. The CTDs also contact DNA adjacent to the CAP site.
Whats a riboswitch?
a regulatory segment of a mRNA that binds to a small molecules that results in a change in protein production
Where does the binding with a riboswitch occur?
The 5’ untranslated region
What do most riboswitches do?
suppress gene expression by blocking either termination of transcription or initiation of translation
Some examples include riboswitched for…
- adenine
- lysine coenzyme B12
- glycine
What do the following mechanisms show?
A= ligand binds to site which changes conformation of terminator hairpin which slows down RNA polymerase and terminates transcription
B= no ligand, RBS is available for ribosome, if ligand around then binds to RNA and blocks the initiation of translation
What does supercoiling of DNA do?
blockes RNA polymerase from gettin in and transcribing gene
allows DNA to fit into cell
What will form in front of the RNA polymerase as it move along the ds circular DNA?
Torsional stress
What will the supercoil structure of DNA repress and how?
It will repress gene expression by prevention proteins from binding to DNA
Whats the role of DNA topoisomerases ?
relieve and increase torsional stress on DNA double helix
What do Topoisomerases introduce?
A nick in one or both strands of DNA and can also reseal the broken ends
Whats a universal issue in organisms dependent on DNA for their genetic material?
torsional stress
Whats topoisomerases essential for?
An essential enzyme in bacteria, fungi, plants and animals
What are the types of Topoisomerase enzymes and their function?
